Quantitative photoconversion analysis of internal molecular dynamics in stress granules and other membraneless organelles in live cells
Quantitative photoconversion analysis of internal molecular dynamics in stress granules and other membraneless organelles in live cells
Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020).
Benzothiazoles/chemistry, Biomolecular Condensates/metabolism, HEK293 Cells, Humans, Molecular Dynamics Simulation, Molecular Probe Techniques/instrumentation, Molecular Probes/chemistry, Optical Imaging/methods, Organelles/metabolism, Proteins/metabolism, Stress Granules/metabolism
100217
Amen, Triana
388dc540-e819-4d07-8f1e-ee0f3949a54b
Kaganovich, Daniel
ebb13f4e-e925-4aef-88e7-ddc25ef52d8f
18 December 2020
Amen, Triana
388dc540-e819-4d07-8f1e-ee0f3949a54b
Kaganovich, Daniel
ebb13f4e-e925-4aef-88e7-ddc25ef52d8f
Amen, Triana and Kaganovich, Daniel
(2020)
Quantitative photoconversion analysis of internal molecular dynamics in stress granules and other membraneless organelles in live cells.
STAR protocols, 1 (3), .
(doi:10.1016/j.xpro.2020.100217).
Abstract
Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020).
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Published date: 18 December 2020
Additional Information:
© 2020 The Author(s).
Keywords:
Benzothiazoles/chemistry, Biomolecular Condensates/metabolism, HEK293 Cells, Humans, Molecular Dynamics Simulation, Molecular Probe Techniques/instrumentation, Molecular Probes/chemistry, Optical Imaging/methods, Organelles/metabolism, Proteins/metabolism, Stress Granules/metabolism
Identifiers
Local EPrints ID: 482108
URI: http://eprints.soton.ac.uk/id/eprint/482108
ISSN: 2666-1667
PURE UUID: cd125a20-b2d0-44b1-9e1c-24920609b674
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Date deposited: 19 Sep 2023 16:40
Last modified: 17 Mar 2024 04:22
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Author:
Triana Amen
Author:
Daniel Kaganovich
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